During the tenure of this project we have shown that plasma membrane calcium ATPase (PMCA) and sodium-calcium exchanger (NCX) are deployed on opposing sides of the osteoblast. NCX, being on the matrix-facing cell surface, is advantageously positioned to direct Ca++ into site of mineralization. This past year we reported that specific inhibition of NCX blocks mineralization of extracellular matrix (Stains and Gay, J. Bone Mineral Res. 16:1434-1443,2001). NCX has recently been found to exist as three distinct isoforms (NCX1, NCX2 and NCX3) and we have shown by RT-PCR that NCX1 and NCX3 are expressed in osteoblasts (Stains et al., J. Cell. Biochem., in press). In Aim One of the present proposal we plan to determine which of the three NCX isoforms is critical for mineralization. Antisense oligonucleotides will be used to knockdown or ablate specific isoform expression. The hypothesis to be tested is that loss of functional NCX3 results in impaired mineralization, whereas ablation ofNCX1 or NCX2 has little effect on mineralization. In Aim Two we focus on the distribution of the NCX isoforms and PMCA in relation to the development of osteoblast polarity. The hypothesis to be tested is that segregation of plasma membrane domains with respect to NCX and PMCA occurs when osteoblasts develop lateral contacts and become mature polarized cells. Understanding the timing of expression of NCX isoforms in relation to expression of lateral adhesion devices (e.g. cadherin-1 1 and gap-junction protein, connexin-43) will provide insight into the cellular basis of mineralization. This will also provide a framework to assess how mineralization may be regulated. NCX is emerging as an important ion-translocating protein in osteoblasts. Derangements (e.g. mutations) in NCX can be predicted to result in an undermineralized skeleton. These studies will disclose novel mechanisms by which bone forming cells carry out mineralization of the extracellular matrix.